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Leishmania infantum belongs to the L. donovani complex, which includes species associated with visceral leishmaniasis. Traditionally, antimonial compounds have served as the primary antiparasitic treatment for all clinical forms of leishmaniasis. However, the global spread of resistance to these compounds has posed a significant challenge in the treatment in some regions. In this study, we aimed to investigate resistance to trivalent sodium stibogluconate in vitro using promastigotes from a wild strain of L. infantum. We compared the growth rates and proteomic profiles of wild-type and resistant line conducting label-free quantitative mass spectrometry-based proteomic analyses. Statistical and bioinformatics analyses were employed to evaluate the significance of protein concentration changes, protein identity annotation, GO term analysis, biosynthetic pathways, and protein-protein interactions. Our findings revealed that the resistant line displayed a notable reduction in growth rate. Proteomic data unveiled similar protein concentrations per cell in both groups but with differing molecule copy numbers. We identified 165 proteins with increased concentration, these were associated with transcription and translation activities, lipid metabolism, energy metabolism, and peroxisome biogenesis. In the decreased protein groups were 56 proteins linked to metal acquisition and metabolism, particularly iron. These results suggest a novel perspective on antimonial resistance, highlighting the importance of post-transcriptional and post-translational regulation, alongside energy expenditure compensation and alterations in organelle membrane lipid composition in antimonial-resistant parasites. Overall, our study provides insights into the proteomic profile of stibogluconate-resistant strain, contributing to our general understanding of the complex landscape of antiparasitic resistance in L. infantum. SIGNIFICANCE: Species within the Leishmania donovani complex are implicated in cases of visceral leishmaniasis in the world. Leishmania infantum is a species that predominates in regions spanning the Mediterranean Basin, the Middle East, Central Asia, South and Central America. Antimonials were the first treatment for leishmaniasis, however in the last decades, the resistance has emerged in subregions like India, where it is not a therapeutic option. In contrast, sodium stibogluconate (SbIII) remains the first-line treatment in the Americas. Unfortunately, the emergence of resistance has outpaced the development of new therapeutic options, thereby becoming a critical point in the struggle against the disease. In this study we performed an in-depth proteomic analysis with liquid chromatography mass-mass spectrometry (LC-MS/MS) on L. infantum with Sb-induced resistance in vitro. Results showed a complex proteomic adaptation in the resistant line, involving transcriptional and translational proteins, energy compensation, and homeostasis maintenance. These insights contribute to understanding the molecular adaptation in the parasite and provide information to new investigations related to therapeutics development., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

The present review article recapitulates for the first time the antipathogenic biological data of a series of lipidic natural products and synthetic analogues thereof characterized by the presence in their structure of an alkynylcarbinol unit. The cytotoxic properties of such natural and bioinspired compounds have been covered by several literature overviews, but to date, no review article detailing their activity against pathogens has been proposed. This article thus aims at providing a comprehensive overview of the field including early studies from the 1970s and 1980s with a specific focus on results published from the late 1990s until nowadays. Publications presenting the data of almost 50 different natural products are reported. Detailed activities encompass the fields of leishmanicidal, antiplasmodial, trypanocidal, fungicidal, and mainly antibacterial and antimycobacterial compounds. The few published studies aimed at exploring the structure-activity relationship in these series are also described. Around 15 different synthetic analogues of natural products, selected among the most active reported, are also presented. The rare data available regarding the antipathogenic mode of action of these products are recalled, and finally, a comparative analysis of the available biological data is proposed with the aim of identifying the key structural determinants for the bioactivity against pathogens of these unusual compounds.

Background: The fabrication of anti-Toxoplasma drugs with less side effects and desirable efficacy is one of the important research goals facing with toxoplasmosis. This study aimed to determine the anti-Toxoplasma effects of Cinnamon zeylanicum (CZ), Moringa oleifera (MO) oil encapsulated into solid lipid nanoparticles (SLNs)., Methods: Vero cells were cultured with serial concentrations (1 mg/mL to 100 µg/mL) of CZ-SLNs and MO-SLNs in DMEM culture medium. The morphological, physical, and chemical features of nanoparticles were calculated. The cell viability assays and anti-T. gondii effects of CZ-SLNs and MO-SLNs were evaluated. The CC 50 and IC 50 indices of SLNs-enveloped extracts were calculated., Results: The particle sizes of MO-SLNs and CZ-SLNs were 411.5 and 365 nm, while PDI indices were 0.53 and 0.7, respectively. Transmission electron microscopy (TEM) showed that both MO-SLNs and CZ-SLNs were smoothed spherical nanoparticles with rounded edges. The cytotoxicity assay showed the CC 50 value of MO-SLNs at concentrations of ˃10 mg/mL. In addition, 60% of T. gondii-infected Vero cells remained alive at the concentrations ≤ 1 mg/ml, while the MO-SLNs killed at least 90% of T. gondii tachyzoites with an IC 50  > 1 µg/ml. The cytotoxicity of CZ-SLNs extract showed the CC 50 at the concentration 0.1 mg/mL. More than 50% of Vero cells, infected with T. gondii tachyzoites, survived at a concentration less than 0.1 mg/mL (IC 50 ˂ 0.1 mg/mL), while the CZ-SLNs killed at least 85% of T. gondii tachyzoites in all concentrations., Conclusion: The current results represented that the use of SLNs as a nano-carrier for M. oleifera and C. zeylanicum could kill T. gondii tachyzoites with low cytotoxicity, suggesting the effectiveness of these nano-emulsions along with the chemical agents in the treatment of Toxoplasma., (© 2024. The Author(s).)

Primary amoebic meningoencephalitis (PAM) is a human brain infection caused by Naegleria fowleri with a 97% mortality rate. Quinazolinones resulting from a Mannich-coupled domino rearrangement were recently identified as inhibitors of the amoeba. Herein, we resolved the effective concentrations for 25 pilot compounds and then, using the Mannich protocol and a key late-stage, N -demethylation/functionalization, we synthesized 53 additional analogs to improve potency, solubility and microsomal stability. We established an antiamoebic quinazolinone pharmacophore, culminating in (±)- trans - 57b which featured the best combination of potency, selectivity index, solubility, and microsomal stability. Enantiomeric separation afforded (4a S ,13b R )- 57b ( BDGR-20237 ) with a 41-fold potency advantage over its enantiomer. ADME and mouse pharmacokinetic profiling for BDGR-20237 revealed high brain penetrance but a limited half-life which did not statistically enhance the mouse survival in a pilot efficacy study. The pharmacophoric model, supported by 88 quinazolinones, several of which exhibit subnanomolar potency, will guide further scaffold optimization.

Macrocycles are prominent among drugs for treatment of infectious disease, with many originating from natural products. Herein we report on the discovery of a series of macrocycles structurally related to the natural product hymenocardine. Members of this series were found to inhibit the growth of Plasmodium falciparum , the parasite responsible for most malaria cases, and of four kinetoplastid parasites. Notably, macrocycles more potent than miltefosine, the only oral drug used for the treatment of the neglected tropical disease visceral leishmaniasis, were identified in a phenotypic screen of Leishmania infantum . In vitro profiling highlighted that potent inhibitors had satisfactory cell permeability with a low efflux ratio, indicating their potential for oral administration, but low solubility and metabolic stability. Analysis of predicted crystal structures suggests that optimization should focus on the reduction of π-π crystal packing interactions to reduce the strong crystalline interactions and improve the solubility of the most potent lead.

Objective: Antimicrobial resistance is a real threat to humanity. Pentavalent antimonials are reported non-effective in leishmaniasis treatment today, in countries like India. New treatment options have been assessed worldwide lately. Antimicrobial peptides (AMP) are the leading antibiotic candidates due to their large spectrum, fast efficacy, and low resistance risks. Cathelicidins are the AMP with well-documented antimicrobial activities against bacteria, fungi, and protozoa, over their positively charged membranes. Here, we aim to design cathelicidine-like helical peptides (CLHP), and compare their anti- Leishmanial efficacies in vitro , with meglumine antimoniate (MA) on Leishmania tropica ., Methods: A total of five study [TN-1-5] and two control (MA and non-drug) groups were formed. Cryopreserved L. tropica isolate was thawed and cultivated in Novy-MacNeal-Nicolle medium and then in RPMI. Five different CLHPs (TN1-5) were diluted in dimethyl sulphoxide. A total of 150 uL of CLHPs and MA were added into the first wells of the test plaques, followed by serial dilutions that revealed doses within 4 and 512 ug/mL. Then, 100 uL of cultures including 1x10 8 /mL of L. tropica promastigotes were added into each well. Viability of promastigotes was checked with XTT, while the parasite count was assessed at 24 th and 48 th hours., Results: TN3 was effective at 32 ug/mL. All tested CLHPs exhibited varying degrees of anti- Leishmanial activities, except TN5, even at its highest dose., Conclusion: TN3 showed a particular efficacy against L. tropica in vitro . Further studies including in vivo testing of the candidate's both efficacy and toxicity are essential., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors.

Indazoles have previously been identified as molecules with antiprotozoal activity. In this study, we evaluate the in vitro activity of thirteen 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) against L. amazonensis , L. infantum, and L. mexicana . In vitro, cytotoxicity against mouse peritoneal macrophages and growth inhibitory activity in promastigotes were evaluated for all compounds, and those showing adequate activity and selectivity were tested against intracellular amastigotes. Transmission and scanning electron microscopy were employed to study the effects of 3-alkoxy-1-benzyl-5-nitroindazole and 2-benzyl-5-nitroindazolin-3-one derivatives on promastigotes of L. amazonensis . Compounds NV6 and NV8 were active in the two life stages of the three species, with the latter showing the best indicators of activity and selectivity. 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) showed in vitro activity comparable to that of amphotericin B against the promastigote stage of Leishmania spp. Two compounds were also found to be active the amastigote stage. Electron microscopy studies confirmed the antileishmanial activity of the indazole derivatives studied and support future research on this family of compounds as antileishmanial agents.

Leishmaniasis is considered one of the most critical health concerns in the world. Unfortunately, no protective vaccines exist and conventional treatments are relatively ineffective. Therefore, new strategies are necessary against leishmaniasis. In recent years, exosomes have shown promising therapeutic outcomes in various diseases, including infectious diseases. In this regard, we aimed to explore the effect of the exosome, pyrimethamine and their combination on the anti-parasitic function of RAW264.7 cells against Leishmania major. Exosomes were isolated from the C57BL/6 peritoneal macrophages. L. major infected and non-infected RAW264.7 cells treated with exosomes, pyrimethamine (PM), and exosomes along with PM. The effect of the treatments was analysed on phagocytosis, efferocytosis, the intracellular parasite count, arginase activity, nitric oxide (NO) and reactive oxygen species (ROS) production. Exosomes could significantly elevate the phagocytosis, efferocytosis, NO and ROS in both infected and non-infected groups (Pv < 0.05). The exosomes reduced the arginase activity in both groups (Pv < 0.05). The intracellular parasite count was significantly lower after treatment with exosomes (Pv < 0.05). These results demonstrate that MQ-derived exosomes can enhance in vitro anti-parasitic responses against L. major. This provides a potential pathway for more effective treatments and underscores the importance of further research in this area., (© 2024 John Wiley & Sons Ltd.)

Leishmaniasis is a parasitic disease spread by the bite of an infected sandfly and caused by protozoan parasites of the genus Leishmania. Currently, there is no vaccine available for leishmaniasis in humans, and the existing chemotherapy methods face various clinical challenges. The majority of drugs are limited to a few toxic compounds, with some parasite strains developing resistance. Therefore, the discovery and development of a new anti-leishmanial compound is crucial. One promising strategy involves the use of nanoparticle delivery systems to accelerate the effectiveness of existing treatments. In this study, Amphotericin B (AmB) was incorporated into functionalized carbon nanotube (f-CNT) and evaluated for its efficacy against Leishmania major in vitro and in a BALB/c mice model. The increase in footpad thickness was measured, and real-time PCR was used to quantify the parasite load post-infection. Levels of nitric oxide and cytokines IL-4 and IFN-γ were also determined. We found that f-CNT-AmB significantly reduced the levels of promastigotes and amastigotes of the Leishmania parasite. The nanoparticle showed strong anti-leishmanial activity with an IC 50 of 0.00494 ± 0.00095 mg/mL for promastigotes and EC 50 of 0.00294 ± 0.00065 mg/mL for amastigotes at 72 h post-infection, without causing harm to mice macrophages. Treatment of infected BALB/c mice with f-CNT-AmB resulted in a significant decrease in cutaneous leishmania (CL) lesion size in the foot pad, as well as reduced Leishmania burden in both lymph nodes and spleen. The levels of nitric oxide and IFN-γ significantly increased in the f-CNT-AmB treated groups. Also, our results showed that the level of IL-4 significantly decreased after f-CNT-AmB treatment in comparison to other groups. In conclusion, our results demonstrate that AmB loaded into f-CNT is significantly more effective than AmB alone in inhibiting parasite propagation and promoting a shift towards a Th1 response., (© 2024 John Wiley & Sons Ltd.)

Miltefosine is the first and only drug approved for the treatment of leishmaniasis. It is also known as a PI3K/AKT signaling pathway inhibitor utilized in anti-cancer or anti-viral therapies. However, the impact of miltefosine on male fertility has not been fully understood. Therefore, this study was performed to investigate the effects of miltefosine on sperm function during capacitation. Duroc spermatozoa were exposed to 0, 2.5, 5, 10, 20, 40, and 80 μM miltefosine and induced for capacitation. Our results showed that miltefosine dramatically increased the expression of PI3K/AKT signaling pathway-associated proteins. Sperm motility, motion kinetics, capacitation, and tyrosine phosphorylation were significantly suppressed by miltefosine. However, intracellular ATP levels and cell viability were not significantly affected. Our findings suggest that miltefosine may disrupt sperm function by abnormally increasing the levels of PI3K/AKT signaling pathway-associated proteins. Therefore, the harmful effects of miltefosine on male reproduction should be considered when using this drug., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protozoal diarrhea caused by Tritrichomonas foetus (blagburni) is a prevalent, lifelong, and globally distributed burden in domestic cats. Treatment is limited to the use of 5-nitroimidazoles and treatment failure is common. The repurposed gold salt compound auranofin has killing activity against diverse protozoa in vitro but evidence of efficacy in naturally occurring protozoal infections is lacking. This exploratory study investigated the efficacy and safety of auranofin for treatment of cats with naturally occurring, 5-nitroimidazole-resistant, T. foetus infection. The minimum lethal concentration (MLC) of auranofin against 5 isolates of feline T. foetus was determined under aerobic conditions in vitro. Healthy cats and cats with T. foetus infection were treated with immediate release auranofin (range, 0.5-3 mg/cat for 7 days) or guar gum-coated auranofin capsules (0.5 or 3 mg/cat for 7 days). Adverse effects were monitored by clinical signs and clinicopathologic testing. Efficacy was determined by fecal consistency score, bowel movement frequency, and single-tube nested PCR of feces for T. foetus rDNA. Fecal samples were assayed for concentrations of auranofin, known and predicted metabolites of auranofin, gold containing molecules, and total gold content using HPLC, LC-MS, ion mobility-MS, and ICP-MS, respectively. Auranofin was effective at killing isolates of feline T. foetus at MLC ≥ 1 μg/ml. Treatment of cats with T. foetus infection with either immediate release auranofin or a colon-targeted guar gum-coated tablet of auranofin did not eradicate infection. Treatment failure occurred despite fecal concentrations of gold that met or exceeded the equivalent MLC of auranofin. Neither auranofin, known or predicted metabolites of auranofin, nor any gold-containing molecules >100 Da could be detected in fecal samples of treated cats. Adverse effects associated with auranofin treatment were common but minor. These studies identify that in vitro susceptibility test results of auranofin may not translate to treatment effectiveness in vivo even when achieving gold concentrations equivalent to the MLC of auranofin in the target environment. These studies further establish the absence of any predicted or unpredicted gold containing metabolites in feces after oral administration of auranofin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Over the past 20 years, significant progress has been made in anti-leishmanial therapy. Three new drugs/formulations are available for the treatment of various forms of leishmaniasis, namely oral miltefosine, paromomycin and liposomal amphotericin B. However, these advances in drug development have added considerable complexity for clinicians including toxicity, emergence of resistance and decreased sensitivity of available drugs. The development of newer drugs with less toxicity and more efficacy is urgently needed., Areas Covered: This review comprehensively examines the latest developments and current status of antileishmanial drugs for the treatment of leishmaniasis across the world. Several new investigational drugs that showed anti-leishmanial activity under in vitro or in vivo conditions and either underwent the phase-I/II clinical trials or are on the verge of entering the trials were reviewed. We also delve into the challenges of drug resistance and discuss the emergence of new and effective antileishmanial compounds., Expert Opinion: The available treatments for leishmaniasis are limited in number, toxic, expensive, and demand extensive healthcare resources. Every available antileishmanial drug is associated with several disadvantages, such as drug resistance and toxicity or high cost. Miltefosine is potentially teratogenic. New antileishmanial drugs/treatment modalities are sorely needed for expanding future treatment options.

Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan that infects warm-blooded animals and has a global distribution. Acute toxoplasmosis is commonly reported in patients with acquired/congenital toxoplasmosis and immune deficiency. New methods are needed to prevent the sideffects of classical treatment. In this study, Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS) were synthesized and zeta potential and size were determined, and an MTT assay was performed to evaluate the cell toxicity on Macrophage cells (MQ) and anti-Toxoplasma activity using Trypan-blue staining by different concentrations of Rosuvastatin (ROS), and Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS). The cell viability assay demonstrated that CH-NP-ROS had lower cell toxicity (<15 %) compared to ROS (<30 %). Statistical analysis showed that CH-NP-ROS significantly killed 98.950 ± 1.344; P < 0.05) of Toxoplasma gondii tachyzoites. In vivo results of perituneal fluid showed that CH-NP significantly reduced the parasite load in the CH-NP-ROS group, compared to that in negative control group (P < 0.001). Growth inhibition rates of tachyzoites in mice receiving free ROS and CH-NP-ROS (injection and oral form) were found to be 166.125 + 4.066, 118.750 + 4.596 and 124.875 + 2.652, respectively, compared to mice in Sulfadiazine/Pyrimethamine treated group (positive control). In the infected untreated mice (control +), the mean tachyzoite counts per oil immersion field in the spleen was 8.25 respectively. The mean survival time in all the groups treated with ROS and CH-NP-ROS was longer than that in the negative control group Therefore, nanoformulation is a promising approach for the delivery and is safe for using therapeutic effects in acute toxoplasmosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC-CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200 µM and mean inhibitory concentration (IC 50 ) values lower than 10 µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 has potential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Cryptosporidium is among the top causes of life-threatening diarrheal infection in public health and livestock sectors. Despite its high prevalence and economic importance, currently, there is no vaccine. Control of this protozoan is difficult due to the excretion of many resistant oocysts in the feces of the infected host, which contaminate the environment. Paromomycin shows inconsistent results and isn't considered a reliable therapy for cryptosporidiosis. Nitazoxanide (NTZ), the only FDA-approved drug against this parasite, is less productive in impoverished children and PLWHA (people living with HIV/AIDS). The absence of mitochondria and apicoplast, its unique location inside enterocytes separated by parasitophorous vacuole, and, most importantly, challenges in its genetic manipulations are some hurdles to the drug-discovery process. A library of compounds has been tested against Cryptosporidium during in vitro and in vivo trials. However, there has still not been sufficient success in finding the drug of choice against this parasite. Recent genome editing technologies based on CRISPR/Cas-9 have explored the functions of the vital genes by producing transgenic parasites that help to screen a collection of compounds to find target-specific drugs, provided the sufficient availability of in vitro culturing platforms, efficient transfection methods, and analytic techniques. The use of herbal remedies against Cryptosporidium is also an emerging area of interest with sufficient clinical success due to enhanced concern regarding anthelmintic resistance. Here, we highlighted present treatment options with their associated limitations, the use of genetic tools and natural products against it to find safe, effective, and inexpensive drugs to control the ever-increasing global burden of this disease., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mexico's Yucatan Peninsula is an endemic area of cutaneous leishmaniasis, locally known as the chiclero's ulcer, and Mayan traditional medicine which refers to the use of Thouinia paucidentata Radlk, known as k'an chuunup. Aqueous and organic leaves extracts were evaluated against promastigotes and amastigotes of Leishmania mexicana. Toxicity tests of extracts were performed using Vero and J774A.1 macrophage cell lines. The composition of the most active extracts was analysed by GC-MS. The n -hexane and ethyl acetate extracts showed potent anti- Leishmania activity against the promastigote form, and remarkably, n -hexane extract exhibited potent activity against the amastigote form. Both extracts showed low toxicity on Vero both not on J774A.1 cells. Analysis of both bioactive extracts identified as more abundant compounds, germacrene D-4-ol and thunbergen in n -hexane, and thunbergol in ethyl acetate extracts. Our study presents T. paucidentata as anti- Leishmania phytomedicine supporting its medicinal use and contributes to the understanding of its phytochemical composition.

Extracts from leaves and stems of Thymelaea tartonraira (L.) All. growing in Tunisia were characterised for the presence of flavonoids and phenolic acids by LC-ESI-MS analysis. Twelve flavonoids and ten phenolic acids were detected for the first time in the aerial parts of this plant species, the most abundant compounds being gallic acid, kaempferol, catechin, chlorogenic acid, naringenin and acacetin. The extracts were subjected to in vitro antileishmanial, antifungal and cytotoxic assays, showing promising antileishmanial activity for the E6 dichloromethane extract from the stems (IC 50 values of 1.12 ± 0.50 and 5.41 ± 1.84 µg/mL on L. donovani axenic and intramacrophagic amastigotes, respectively) at the level of the reference drug miltefosine for axenic model. No antifungal activity was observed against Candida albicans (CAAL) and Aspergillus fumigatus (ASFU) strains, with the exception of the E6 dichloromethane extract (IC 50 value of 25.28 ± 4.89 µg/mL on CAAL93 strain). Low toxicity was also highlighted against macrophages Raw 264.7 cells. These promising results point out Thymelaea tartonraira (L.) All. extracts as a valuable source of new natural products to combat leishmaniasis.

Cryptosporidiosis, a prevalent gastrointestinal illness worldwide, is caused by the protozoan parasite Cryptosporidium parvum. Calcium-dependent protein kinase 1 (CpCDPK1), crucial for the parasite's life cycle, serves as a promising drug target due to its role in regulating invasion and egress from host cells. While potent Pyrazolopyrimidine analogs have been identified as candidate hit molecules, they exhibit limitations in inhibiting Cryptosporidium growth in cell culture, prompting exploration of alternative scaffolds. Leveraging the most potent compound, RM-1-95, co-crystallized with CpCDPK1, an E-pharmacophore model was generated and validated alongside a deep learning model trained on known CpCDPK1 compounds. These models facilitated screening Enamine's 2 million HTS compound library for novel CpCDPK1 inhibitors. Subsequent hierarchical docking prioritized hits, with final selections subjected to Quantum polarized docking for accurate ranking. Results from docking studies and MD simulations highlighted similarities in interactions between the cocrystallized ligand RM-1-95 and identified hit molecules, indicating comparable inhibitory potential against CpCDPK1. Furthermore, assessing metabolic stability through Cytochrome 450 site of metabolism prediction offered crucial insights for drug design, optimization, and regulatory approval processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The present study is focused on the isolation and identification of new therapeutic candidates from Chrysanthellum americanum Vatke., and their efficacy against pteridine reductase-1 (PTR1), a valid chemotherapeutic target in the Leishmania parasite. Henceforth, a new compound, chrysanamerine (1), along with 7 known compounds, polyacetylene 2, and flavonoids 3-8, were isolated from C. americanum. Their structures were determined by chemical and spectroscopic analyses and compared with the reported spectroscopic data. All compounds were evaluated for their anti-leishmanial activity against PTR1 via biochemical mechanism-based assay. The in vitro results showed five potential hits including a new compound, chrysanamerine (1), and four known compounds against the PTR1 enzyme. Among them, compound 1 showed a potent enzyme inhibition with an IC 50 of 31.02 ± 2.36 μM, whereas a moderate inhibition was observed in cases of compounds 5 and 6 (IC 50  = 59.86 ± 3.32, and 45.32 ± 3.5 μM, respectively). Whereas, compounds 3 and 8 showed mild inhibition (IC 50  = 72.12 ± 1.12, and 97.18 ± 1.23 μM, respectively) against PTR1, compared with trimethoprim (positive control) (IC 50  = 21.07 ± 1.6 μM). Moreover, the results were further validated via molecular docking and molecular dynamics (MD) simulations. Compound 1 showed a strong affinity to the binding site with a docking score of -11.83, along with the formation of a stable protein-ligand complex over the trajectory of 100 ns. Besides, compounds 1-8 were found to be non-cytotoxic on BJ (human fibroblast) cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Geraniol, a primary component of several essential oils, has been associated with broad-spectrum antiprotozoal activities, although moderate to weak. This study primarily concentrated on the synthesis of hydrazinated geraniol derivatives as potential antiprotozoal agents. The synthesised compounds were tested in vitro against different parasitic protozoans of clinical relevance, including Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania infantum. Compounds 6, 8, 13, 14 and 15 demonstrated low micromolar activity against the different parasites. Compounds 8, 13, 14 and 15 had the highest efficacy against Trypanosoma brucei rhodesiense, as indicated by their respective IC 50 values of 0.74, 0.56, 1.26 and 1.00 µM. Compounds 6, 14 and 15 displayed the best activity against Trypanosoma brucei brucei, with IC 50 values of 1.49, 1.48 and 1.85 µM, respectively. The activity of compounds 6, 14 and 15 also extended to intracellular Trypanosoma cruzi, with IC 50 values of 5.14, 6.30 and 4.90 µM, respectively. Compound 6, with an IC 50 value of 11.73 µM, and compound 14, with an IC 50 value of 8.14 µM, demonstrated some modest antileishmanial activity., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

DNA topoisomerase I (Topo I) is a ubiquitous enzyme that plays a crucial role in resolving the topological constraints of supercoiled DNA during various cellular activities, including repair, replication, recombination, transcription, and chromatin remodeling. Multiple studies have confirmed the essential role of Topo I in nucleic acid metabolism of Leishmania donovani , the kinetoplastid parasite responsible for visceral leishmaniasis or kala-azar. Inhibition of this enzyme has shown promise as a strategy for therapy against visceral leishmaniasis. However, current treatment options suffer from limitations related to effectiveness, cost, and side effects. To address these challenges, computational methods have been employed in this study to investigate the inhibition of Leishmania donovani DNA topoisomerase I (LdTopo I) by phytochemicals derived from Indian medicinal plants known for their anti-leishmanial activity. A library of phytochemicals and known inhibitors was assembled, and virtual screening based on docking binding affinities was conducted to identify potent phytochemical inhibitors. To assess the drug-likeness of the docked phytochemicals, their physicochemical properties were predicted. Additionally, molecular dynamics (MD) simulations were performed on the docked complexes for a duration of 100 ns to evaluate their stability, intermolecular interactions, and dynamic behavior. Among all the docked phytochemicals, three compounds, namely CID23266147 (withanolide N), CID5488537 (fagopyrine), and CID100947536 (isozeylanone), exhibited the highest inhibitory potential against LdTopo I. These findings hold promise for the development of novel inhibitors targeting LdTopo I, which could potentially lead to improved therapies for visceral leishmaniasis.Communicated by Ramaswamy H. Sarma.

Toxoplasmosis is a pervasive parasitic infection possessing a chief impact on both public health and veterinary medicine. Unfortunately, the commercially-available anti-Toxoplasma agents have either serious side effects or diminished efficiency, specifically on the Toxoplasma tissue cysts. In the present study, metformin (The first-line treatment for type 2 diabetes mellitus) was investigated for the first time against chronic cerebral toxoplasmosis in mice model experimentally-infected with ME49 strain versus spiramycin. Two metformin regimens were applied; starting one week before the infection and four weeks PI. Parasitological, ultrastructural, histopathological, immunohistochemical, immunological, and biochemical assessments were performed. The anti-parasitic effect of metformin was granted by the statistically-significant reduction in tissue-cyst burden in both treatment regimens. This was accompanied by markedly-mutilated ultrastructure and profound amelioration of the cerebral histopathology with remarkable decline in the brain CD4 + and CD8 + T cell count. Besides, diminution of anti-Toxoplasma IgG and brain GSH levels was evident. Ultimately, the present findings highlighted the powerful promising therapeutic role of metformin in the management of chronic toxoplasmosis on a basis of anti-parasitic, anti-inflammatory, and anti-oxidant possessions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The methanolic extract of the marine sponge Hemimycale sp . yielded two new compounds; 1-(2'-methyl heptadecyl) phenol ( 1 ) and a new pyrazole derivative; 4-(hydroxymethyl)-1 H -pyrazol-3-ol ( 2 ), together with previously isolated (2' R )-2'-hydroxy-N-((2 S ,3 S ,4 R )-1,3,4-trihydroxy-16-methylpentadecan-2-yl)docosanamide ( 3 ), cholesterol ( 4 ), 5, 8- epi- dioxycholest-6-en-3-ol ( 5 ) and 3-acetylsesterstatin 3 ( 6 ), which were firstly reported from family Hymedesmiidae. Their structure elucidation was based on extensive nuclear magnetic resonance spectroscopy and high resolution-electrospray ionization-mass spectrometry. The isolated compounds were evaluated for their anti-leishmanial and cytotoxic activities. Compound 5 showed remarkable anti-leishmanial activity with IC 50 value of 15.8 ± 0.92 µg/mL comparable with the standard miltefosine (IC 50 = 3.2 ± 0.07 µg/mL), while compound 3 exhibited noteworthy cytotoxicity against A594 cell line with IC 50 value of 29.6 ± 1.68 µg/mL compared to etoposide (IC 50 = 10.9 ± 1.30 µg/mL).

Cryptosporidium parvum (C. parvum), a protozoan parasite, is known to induce significant gastrointestinal disease in humans. Lactate dehydrogenase (LDH), a protein of C. parvum, has been identified as a potential therapeutic target for developing effective drugs against infection. This study utilized a computational drug discovery approach to identify potential drug molecules against the LDH protein of C. parvum. In the present investigation, we conducted a structure-based virtual screening of 55 phytochemicals from the Syzygium aromaticum (S. aromaticum). This process identified four phytochemicals, including Gallotannin 23, Eugeniin, Strictinin, and Ellagitannin, that demonstrated significant binding affinity and dynamic stability with LDH protein. Interestingly, these four compounds have been documented to possess antibacterial, antiviral, anti-inflammatory, and antioxidant properties. The docked complexes were simulated for 100 ns using Desmond to check the dynamic stability. Finally, the free binding energy was computed from the last 10ns MD trajectories. Gallotannin 23 and Ellagitannin exhibited considerable binding affinity and stability with the target protein among all four phytochemicals. These findings suggest that these predicted phytochemicals from S. aromaticum could be further explored as potential hit candidates for developing effective drugs against C. parvum infection. The in vitro and in vivo experimental validation is still required to confirm their efficacy and safety as LDH inhibitors., Competing Interests: Declaration of competing interest The author(s) declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Leishmaniasis is a disease caused by the parasite Leishmania donovani affecting populations belonging to developing countries. The present study explores drug repurposing as an innovative strategy to identify new uses for approved clinical drugs, reducing the time and cost required for drug discovery. The three-dimensional structure of Leishmania donovani Sterol C-24 methyltransferase (LdSMT) was modeled and 1615 FDA-approved drugs from the ZINC database were computationally screened to identify the potent leads. Fulvestrant, docetaxel, indocyanine green, and iohexol were shortlisted as potential leads with the highest binding affinity and fitness scores for the concerned pathogenic receptor. Molecular dynamic simulation studies showed that the macromolecular complexes of indocyanine green and iohexol with LdSMT remained stable throughout the simulation and can be further evaluated experimentally for developing an effective drug. The proposed leads have further demonstrated promising safety profiles during cytotoxicity analysis on the J774.A1 macrophage cell line. Mechanistic analysis with these two drugs also revealed significant morphological alterations in the parasite, along with reduced intracellular parasitic load. Overall, this study demonstrates the potential of drug repurposing in identifying new treatments for leishmaniasis and other diseases affecting developing countries, highlighting the importance of considering approved clinical drugs for new applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to develop microemulsions (MEs) containing α-bisabolol for the topical treatment of cutaneous leishmaniasis (CL). Initially, pseudoternary phase diagrams were developed using α-bisabolol as the oil phase, Eumulgin® CO 40 as the surfactant, Polymol® HE as the co-surfactant, and distilled water as the aqueous phase. Two transparent liquid systems (TLS) containing 5% of α-bisabolol were selected and characterized (F5E25 and F5EP25). Next, skin permeation and retention assays were performed using Franz cells. The interaction of the formulation with the stratum corneum (SC) was evaluated using the FTIR technique. The cytotoxicity was evaluated in murine peritoneal macrophages. Finally, the antileishmanial activity of microemulsions was determined in promastigotes and amastigotes of L. amazonensis (strain MHOM/BR/77/LTB 0016). As a result, the selected formulations showed isotropy, nanometric size (below 25 nm), Newtonian behavior and pH ranging from 6.5 to 6.9. The MEs achieved a 2.5-fold increase in the flux and skin-permeated amount of α-bisabolol. ATR-FTIR results showed that microemulsions promoted fluidization and extraction of lipids and proteins of the stratum corneum, increasing the diffusion coefficient and partition coefficient of the drug in the skin. Additionally, F5E25 and F5EP25 showed higher activity against promastigotes (IC 50 13.27 and 18.29, respectively) compared to unencapsulated α-bisabolol (IC 50 53.8). Furthermore, F5E25 and F5EP25 also showed antileishmanial activity against intracellular amastigotes of L. amazonensis, with IC 50 50 times lower than free α-bisabolol and high selectivity index (up to 15). Therefore, the systems obtained are favorable to topical administration, with significant antileishmanial activity against L. amazonensis promastigotes and amastigotes, being a promising system for future in vivo trials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Leishmaniasis is a widespread parasitic disease and is one of the major public health concerns in developing countries. Many drugs have been identified for leishmania as targets, but the potential toxicity and long-term treatment remain the most significant problems in terms of further development. The present study employed physicochemical investigations, structure-based virtual screening, ADMET analysis, molecular dynamics simulation, and MM-PBSA, to identify potential compounds for Leishmania. We evaluated 30,926 natural products from the NPASS database, and four potentials passed the pharmacokinetic ADMET studies and were verified using the molecular docking approach. Molecular docking results showed good binding interaction of the compounds with the active site of leishmania pteridine reductase enzyme PTR1, with compound TTC1 showing FRED and Autodock binding energies of -10.33 and -10.94, respectively, which were comparable with the antileishmania drugs of Allopurinol, Miltefosine and the original ligand, methotrexate. TTC1 compound was found to be favorable for hydrophobic interaction with PTR1. In addition, the physicochemical properties of the compounds were studied using the SwissADME web server. All compounds followed Lipinski's rule of five and can be considered as good oral candidates. The analysis of the 100 ns molecular dynamics simulation results based on the best-docked TTC1 with PTR1 receptor demonstrates stable interactions, and the complex undergoes low conformational fluctuations. The average of the calculated binding free energy of the TTC1-1e7w complex is (-68.67 kJ/mol), and the result demonstrated that the TTC1 promoted stability to the Leishmania-PTR1 complex. The potential compounds can be further explored for their antileishmanial activity.Communicated by Ramaswamy H. Sarma.

Visceral leishmaniasis (VL), caused by protozoa of the genus Leishmania , remains a significant public health concern due to its potentially lethal nature if untreated. Current chemotherapy options are limited by severe toxicity and drug resistance. Derivatives of 1,2,4-oxadiazole have emerged as promising drug candidates due to their broad biological activity. This study investigated the effects of novel 1,2,4-oxadiazole derivatives ( Ox1 - Ox7 ) on Leishmania infantum , the etiological agent of VL. In silico predictions using SwissADME suggest that these compounds have high oral absorption and good bioavailability. Among them, Ox1 showed the most promise, with higher selectivity against promastigotes and lower cytotoxicity towards L929 fibroblasts and J774.G8 macrophages. Ox1 exhibited selectivity indices of 18.7 and 61.7 against L. infantum promastigotes and amastigotes, respectively, compared to peritoneal macrophages. Ultrastructural analyses revealed severe morphological damage in both parasite forms, leading to cell death. Additionally, Ox1 decreased the mitochondrial membrane potential in promastigotes, as shown by flow cytometry. Molecular docking and dynamic simulations indicated a strong affinity of Ox1 for the L. infantum CYP51 enzyme. Overall, Ox1 is a promising and effective compound against L. infantum .

Drug repurposing is a promising approach towards the discovery of novel treatments against Neglected Tropical Diseases, such as Leishmaniases, presenting the advantage of reducing both costs and duration of the drug discovery process. In previous work, our group developed a Machine Learning pipeline for the repurposing of FDA-approved drugs against Leishmania parasites. The present study is focused on an in vitro validation of this approach by assessing the antileishmanial effects of 10 predicted drug candidates. First, we evaluated the drugs' activity against promastigotes from two strains of L. infantum and one of L. major , which caused distinct clinical manifestations, using an MTT assay. The standard anti- Leishmania drug Amphotericin B was used as a positive control. Five molecules demonstrated anti- Leishmania effects, out of which Acebutolol, Prilocaine and Phenylephrine are described herein for the first time. When tested on promastigote growth, Acebutolol displayed IC 50 values ranging from 69.28 to 145.53 µg/mL. Prilocaine exhibited IC 50 values between 33.10 and 45.81 µg/mL. Phenylephrine, on the other hand, presented IC 50 values >200 µg/mL. The two remaining drugs, Dibucaine and Domperidone, exhibited significantly low IC 50 values varying between 0.58 and 1.05 µg/mL, and 6.30 and 8.17 µg/mL, respectively. Both compounds were previously described as anti- Leishmania agents in vivo . All five compounds demonstrated no notable cytotoxic effects on THP-1-derived macrophages at the IC 50 concentrations, allowing for their testing on the intracellular form of L. major and L. infantum parasites. Interestingly, all compounds exhibited antileishmanial activity on amastigotes with enhanced IC 50 values compared to the corresponding promastigotes. Noticeably, Dibucaine and Domperidone displayed IC 50 values of at most 1.99 µg/mL. Acebutolol, Prilocaine and Phenylephrine showed IC 50 values ranging from 13.84 to 66.81 µg/mL. Our previously published Computer-Aided repositioning pipelines of FDA-approved drugs as antileishmanial agents identified Dibucaine and Domperidone as candidates in support of previous in vivo studies. This study consolidates such findings through the in vitro validation against 2 Leishmania species, highly prevalent in Africa and Middle East, and reveals Acebutolol, Prilocaine, and Phenylephrine as novel anti- Leishmania effectors, confirming the relevance of our approach and calling for further investigations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Oualha, Abdelkrim, Guizani and Harigua-Souiai.)

Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). Entamoeba histolytica , the protozoan parasite responsible for amebiasis, apparently lacks a gene encoding FADS that share similarity with bacterial and eukaryotic canonical FADS, yet it can synthesize FAD. In this study, we have identified the gene responsible for FADS and thoroughly characterized physiological and biochemical properties of FADS from E. histolytica . Phylogenetic analysis revealed that the gene was likely laterally transferred from archaea. The kinetic properties of recombinant EhFADS were consistent with the notion that EhFADS is of archaeal origin, exhibiting K M and k cat values similar to those of the arachaeal enzyme while significantly differing from the human counterpart. Repression of gene expression of EhFADS by epigenetic gene silencing caused substantial reduction in FAD levels and parasite growth, underscoring the importance of EhFADS for the parasite. Furthermore, we demonstrated that EhFADS gene silencing reduced thioredoxin reductase activity, which requires FAD as a cofactor and makes the ameba more susceptible to metronidazole. In summary, this study unveils unique evolutionary and biochemical features of EhFADS and underscores its significance as a promising drug target in combating human amebiasis.IMPORTANCEFAD is important for all forms of life, yet its role and metabolism are still poorly studied in E. histolytica , the protozoan parasite causing human amebiasis. Our study uncovers the evolutionary unique key enzyme, archaeal-type FADS for FAD biosynthesis from E. histolytica for the first time. Additionally, we showed the essentiality of this enzyme for parasite survival, highlighting its potential as target for drug development against E. histolytica infections., Competing Interests: The authors declare no conflict of interest.

Leishmaniasis is a complex vector-borne disease caused by intracellular protozoan parasites of the Leishmania genus. It presents a significant public health challenge in tropical and subtropical regions globally. As resistance to treatment increases, managing and controlling Leishmaniasis becomes more challenging, necessitating innovative approaches. To address this challenge, our study utilized subtractive genomics and structure-based approaches to identify common drug targets and combat antimicrobial resistance (AMR) across five Leishmania species strains. The subtractive genomics approach unraveled Glutamate Dehydrogenase (GDH) as a promising drug target for treating Leishmania infections. The investigation considered established methodologies observed in analogous studies, orthologous group, and druggability tests. Multiple sequence alignment revealed conserved sequences in GDH, while phylogenetic tree analysis provided insights into the evolutionary origin and close relationships of GDH across Leishmania species. Conserved sequences in GDH along with its function in pathogenicity provided insights into the close relationships of GDH across Leishmania species. Using a structure-based approach, our study showed the molecular interactions between GDH and three ligands-Bithionol, GW5074, and Hexachlorophene-through molecular docking and 100 ns molecular dynamics (MD) simulations. GW5074 exhibited a significant affinity for GDH, as indicated by stable RMSD values, a more compact conformation, and a higher number of hydrogen bonds than Bithionol. MMPBSA analysis confirmed the superior binding energy of the GW5074-GDH complex, emphasizing its potential as a potent ligand for drug development. This comprehensive analysis identified GW5074 as a promising candidate for inhibiting GDH activities in Leishmania species, contributing to the development of effective therapeutics against Leishmania infections., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Intracellular infections are difficult to treat, as pathogens can take advantage of intracellular hiding, evade the immune system, and persist and multiply in host cells. One such intracellular parasite, Leishmania , is the causative agent of leishmaniasis, a neglected tropical disease (NTD), which disproportionately affects the world's most economically disadvantaged. Existing treatments have relied mostly on chemotherapeutic compounds that are becoming increasingly ineffective due to drug resistance, while the development of new therapeutics has been challenging due to the variety of clinical manifestations caused by different Leishmania species. The antimicrobial peptide melittin has been shown to be effective in vitro against a broad spectrum of Leishmania , including species that cause the most common form, cutaneous leishmaniasis, and the most deadly, visceral leishmaniasis. However, melittin's high hemolytic and cytotoxic activity toward host cells has limited its potential for clinical translation. Herein, we report a design strategy for producing a melittin-containing antileishmanial agent that not only enhances melittin's leishmanicidal potency but also abrogates its hemolytic and cytotoxic activity. This therapeutic construct can be directly produced in bacteria, significantly reducing its production cost critical for a NTD therapeutic. The designed melittin-containing fusion crystal incorporates a bioresponsive cathepsin linker that enables it to specifically release melittin in the phagolysosome of infected macrophages. Significantly, this targeted approach has been demonstrated to be efficacious in treating macrophages infected with L. amazonensis and L. donovani in cell-based models and in the corresponding cutaneous and visceral mouse models.

Leishmaniasis, attributed to the protozoan parasite Leishmania , manifests in diverse clinical forms, including cutaneous, mucocutaneous, and visceral leishmaniasis; VL constitutes a significant global health menace. Prevalent in tropical and subtropical regions, this affliction disproportionately impacts individuals below the poverty threshold, transmitted through the bite of female sandflies. Existing treatments, such as pentavalent antimony, miltefosine, and Amphotericin B, exhibit limitations. Despite the emergence of liposomal Amphotericin B (AmBisome) as a promising antileishmanial agent, its utility is impeded by adverse effects, elevated production expenses, and cytotoxicity. To address these challenges, our investigation introduces a potential remedy─a citrate-coated gold Amphotericin B nanoparticle formulation. Characterized using dynamic light scattering and transmission electron microscopy, this pioneering formulation exhibited efficacy against L. donovani Ag83 promastigotes as demonstrated by MTT cell viability testing. Evaluating internal reactive oxygen species (ROS) levels and dual staining with acridine orange and ethidium bromide unveiled its consequential impact on cell death. Significantly, our study discloses this novel nanoformulation's unprecedented inhibition of the trypanothione reductase enzyme. The findings posit the citrate-coated gold Amphotericin B nanoformulation as a promising and targeted antileishmanial agent, representing potential advancements in leishmaniasis therapeutics.

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for continued propagation of neglected tropical diseases such as African sleeping sickness, Chagas disease and leishmaniasis respectively. Following a report that captopril targets Leishmania donovani dipeptidyl carboxypeptidase, a series of simple proline amides and captopril analogues were synthesized and found to exhibit 1-2 μM in vitro inhibition and selectivity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. The results were corroborated with computational docking studies. Arguably, the synthetic proline amides represent the structurally simplest examples of in vitro pan antiprotozoal compounds., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Terry K. Smith reports financial support was provided by UKRI Medical Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, we present the design, synthesis, and cytotoxic evaluation of a series of benzimidazole N-acylhydrazones against strains of T. cruzi (Y and Tulahuen) and Leishmania species (L. amazonensis and L. infantum). Compound (E)-N'-((5-Nitrofuran-2-yl)methylene)-1H-benzo[d]imidazole-2-carbohydrazide demonstrated significant activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC 50 /120 h of 0.033 μM and a selectivity index (SI) of 7680. This represents a potency 46 times greater than that of benznidazole (IC 50 /120 h = 1.520 μM, SI = 1390). Another compound (E)-N'-(2-Hydroxybenzylidene)-1H-benzo[d]imidazole-2-carbohydrazide showed promising activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC 50 /120 h of 3.600 μM and an SI of 14.70. However, its efficacy against L. infantum and L. amazonensis was comparatively lower. These findings provide valuable insights for the development of more effective treatments against Trypanosoma cruzi., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recent efforts in the study of vector-borne parasitic diseases (VBPDs) have emphasized an increased consideration for preventing drug resistance and promoting the environmental safety of drugs, from the beginning of the drug discovery pipeline. The intensive use of the few available antileishmanial drugs has led to the spreading of hyper-resistant Leishmania infantum strains, resulting in a chronic burden of the disease. In the present work, we have investigated the biochemical mechanisms of resistance to antimonials, paromomycin, and miltefosine in three drug-resistant parasitic strains from human clinical isolates, using a whole-cell mass spectrometry proteomics approach. We identified 14 differentially expressed proteins that were validated with their transcripts. Next, we employed functional association networks to identify parasite-specific proteins as potential targets for novel drug discovery studies. We used SeqAPASS analysis to predict susceptibility based on the evolutionary conservation of protein drug targets across species. MATH-domain-containing protein, adenosine triphosphate (ATP)-binding cassette B2, histone H4, calpain-like cysteine peptidase, and trypanothione reductase emerged as top candidates. Overall, this work identifies new biological targets for designing drugs to prevent the development of Leishmania drug resistance, while aligning with One Health principles that emphasize the interconnected health of people, animals, and ecosystems.

Primary amoebic meningoencephalitis is caused by the free-living amoeba Naegleria fowleri . The lack of standardized treatment has significantly contributed to the high fatality rates observed in reported cases. Therefore, this study aims to explore the anti- Naegleria activity of eight synthesized cyanoacrylamides and 5-iminopyrrol-2-ones. Notably, QOET-109, QOET-111, QOET-112, and QOET-114 exhibited a higher selectivity index against Naegleria compared to those of the rest of the compounds. Subsequently, these chemicals were assessed against the resistant stage of N. fowleri , demonstrating activity similar to that observed in the vegetative stage. Moreover, characteristic events of programmed cell death were evidenced, including chromatin condensation, increased plasma membrane permeability, mitochondrial damage, and heightened oxidative stress, among others. Finally, this research demonstrated the in vitro activity of the cyanoacrylamide and 5-iminopyrrol-2-one molecules, as well as the induction of metabolic event characteristics of regulated cell death in Naegleria fowleri .

Recently, the growth of consumer demand for functional foods with potential nutritional and health benefits led to rapid growth of analytical tools for profiling of bioactive metabolites and assure quality. Bee propolis is one of the most important bee products owing to its myriad health value. As a gummy exudate produced in beehives after harvesting from different plant species, bee propolis contains bioactive secondary metabolites. The current study aims to profiling the chemical composition of propolis samples from Nigeria using HPLC-UV-ELSD and with the aid of NMR-based analysis for assignment of metabolites classes abundant in Nigerian propolis. Red Nigerian propolis samples were subjected to phytochemical analysis using HPLC-UV-ELSD and NMR. Further chromatographic separation of promising fractions was performed by column chromatography and size exclusion chromatography. Screening of the antitrypanosomal and cytotoxic activities against Trypanosoma brucei and human leukemia cell lines (U937), respectively, was performed. The performance of LC-MS permitted identification of the different components from which 13 compound were identified and allowed combination of fractions to afford 9 fractions from which two isoflavonoids were isolated and identified using 1D and 2D NMR analysis with MS as isosativan and Medicarpin. Red Nigerian propolis crude extract showed the highest inhibitory activity at 6.5 µg/ml compared to moderate activity for the isolated compounds with MIC of 7.6 µg/ml and 12.1 µg/ml for medicarpin and isosativan, respectively. Moreover, the fraction RN-6 from the total extract showed the potent cytotoxic effect with IC 50  = 26.5 µg/ml compared to standard diminazen which showed IC 50  = 29.5 µg/ml., (© 2024. The Author(s).)

Cutaneous leishmaniasis, caused by Leishmania parasites, requires treatments with fewer side effects than those currently available. The development of a topical solution based on amphotericin B (AmB) was pursued. The considerable interest in deep eutectic solvents (DESs) and their remarkable advantages inspired the search for a suitable hydrophobic excipient. Various mixtures based on commonly used hydrogen bond donors (HBDs) and acceptors (HBAs) for DES preparations were explored. Initial physical and in-vitro screenings showed the potential of quaternary phosphonium salt-based mixtures. Through thermal analysis, it was determined that most of these mixtures did not exhibit eutectic behavior. X-ray scattering studies revealed a sponge-like nanoscale structure. The most promising formulation, based on a combination of trihexyl(tetradecyl)phosphonium chloride and 1-oleoyl-rac-glycerol, showed no deleterious effects through histological evaluation. AmB was fully solubilized at concentrations between 0.5 and 0.8 mg·mL -1 , depending on the formulation. The monomeric state of AmB was observed by circular dichroism. In-vitro irritation tests demonstrated acceptable viability for AmB-based formulations up to 0.5 mg·mL -1 . Additionally, an ex-vivo penetration study on pig ear skin revealed no transcutaneous passage, confirming AmB retention in healthy, unaffected skin., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: François-Xavier LEGRAND reports financial support was provided by French National Research Agency. Andras WACHA reports financial support was provided by Hungarian National Research, Development and Innovation Office. Andraas WACHA reports financial support was provided by Hungarian Academy of Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Leishmaniases, a group of neglected tropical diseases caused by an intracellular parasite of the genus Leishmania, have significant impacts on global health. Current treatment options are limited due to drug resistance, toxicity, and high cost. This study aimed to develop nanostructured lipid carriers (NLCs) for delivering Citrus sinensis essential oil (CSEO) and its main constituent, R-limonene, against leishmaniasis. The influence of surface-modified NLCs using chitosan was also examined. The NLCs were prepared using a warm microemulsion method, and surface modification with chitosan was achieved through electrostatic interaction. These nanocarriers were characterized by differential scanning calorimetry (DSC), X-ray diffraction (XRD), transmission electron microscopy, and dynamic light scattering (DLS). In vitro cytotoxicity was assessed in L929 and RAW 264.7 cells, and leishmanicidal activity was evaluated against promastigote and amastigote forms. The NLCs were spherical, with particle sizes ranging from 97.9 nm to 111.3 nm. Chitosan-coated NLCs had a positive surface charge, with zeta potential values ranging from 45.8 mV to 59.0 mV. Exposure of L929 cells to NLCs resulted in over 70 % cell viability. Conversely, surface modification significantly reduced the viability of promastigotes (93 %) compared to free compounds. Moreover, chitosan-coated NLCs presented a better IC 50 against the amastigote forms than uncoated NLCs. Taken together, these findings demonstrate the feasibility of using NLCs to overcome the limitations of current leishmaniasis treatments, warranting further research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Toxoplasmosis, caused by Toxoplasma gondii , poses serious health issues for humans and animals. Individuals with impaired immune systems are more susceptible to severe toxoplasmosis. Pregnant women infected by T. gondii can face the possibility of birth defects and miscarriages. While pyrimethamine and sulfadiazine are commonly used drugs in clinical practice, concerns over their side effects and resistance are on the rise. A spider peptide XYP1 isolated from Lycosa coelestis had potent anti-T. gondii effects, but it had a high synthesis cost and strong cytotoxicity., Methods: This study intended to modify XYP1 for producing derived peptides via amino acid truncation and substitution. The anti-T. gondii effect was evaluated by trypan blue staining assay and killing experiment of RH strain tachyzoites. The CCK8 and hemolysis assays were used to compare their safeties. The morphological changes of T. gondii were observed by scanning electron microscope and transmission electron microscope. In addition, the mechanism of XYP1 against T. gondii through RNA-sequencing was further explored., Results: In vivo and in vitro experiments revealed that XYP1-18 and XYP1-18-1 had excellent anti-T. gondii activity with lower cytotoxicity and hemolysis activity than XYP1. XYP1, XYP1-18, and XYP1-18-1 were able to disrupt the surface membrane integrity of T. gondii tachyzoites, forming pores and causing the disruption of organelles. Furthermore, RNA-sequencing analysis indicated that XYP1 could stimulate the host immune response to effectively eliminate T. gondii and lessen the host's inflammatory reaction., Conclusions: XYP1-18 had lower cytotoxicity and hemolysis activity than XYP1, as well as significantly extending the survival time of the mice. XYP1 played a role in host inflammation and immune responses, revealing its potential mechanism. Our research provided valuable insights into the development and application of peptide-based drugs, offering novel strategies and directions for treating toxoplasmosis., (© 2024. The Author(s).)

Background: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms., Objectives: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development., Methods: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays., Results and Discussion: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50 > 100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTm = 1 ± 0.1°C., Conclusions: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: Leishmaniasis is a neglected zoonosis caused by parasites of Leishmania spp. The main drug used to treat cutaneous leishmaniasis (CL) is the antimoniate of meglumine. This drug, which has strong adverse and toxic effects, is usually administered intravenously, further complicating the difficult treatment. Factors such as Leishmania gene expression and genomic mutations appear to play a role in the development of drug resistance., Objectives: This systematic review summarises the results of the literature evaluating parasite genetic markers possibly associated with resistance to pentavalent antimony in CL., Methods: This study followed PRISMA guidelines and included articles from PubMed, SciELO, and LILACS databases. Inclusion criteria were studies that (i) investigated mutations in the genome and/or changes in gene expression of Leishmania associated with treatment resistance; (ii) used antimony drugs in the therapy of CL; (iii) used naturally resistant strains isolated from patients. The Joanna Briggs Institute Critical Appraisal Checklist was used to assess article quality and risk of bias., Findings: A total of 23 articles were selected, of which 18 investigated gene expression and nine genomic mutations. Of these 23 articles, four examined gene expression and genomic mutations in the same samples. Regarding gene expression, genes from the ABC transporter protein family, AQP1, MRPA, TDR1 and TRYR were most frequently associated with drug resistance. In one of the articles in which mutations were investigated, a mutation was found in HSP70 (T579A) and in three articles mutations were found in AQP1 (A516C, G562A and G700A). A limitation of this review is that in most of the included studies, parasites were isolated from cultured lesion samples and drug resistance was assessed using in vitro drug susceptibility testing. These approaches may not be ideal for accurate genetic evaluation and detection of treatment failure., Main Conclusions: The development of further studies to evaluate the genetic resistance factors of Leishmania spp. is necessary to elucidate the mechanisms of the parasite and improve patient treatment and infection control.

Background: Leishmaniasis is a deadly protozoan parasitic disease and a significant health problem in underdeveloped and developing countries. The global spread of the parasite, coupled with the emergence of drug resistance and severe side effects associated with existing treatments, has necessitated the identification of new and potential drugs., Objective: This study aimed to identify promising compounds for the treatment of leishmaniasis by targeting two essential enzymes of Leishmania donovani: trypanothione reductase (Try-R) and trypanothione synthetase (Try-S)., Methods: High-throughput virtual and in vitro screening of in-house and commercial databases was conducted. A pharmacophore model with seven features was developed and validated using the Guner-Henery method. The pharmacophore-based virtual screening yielded 690 hits, which were further filtered through Lipinski's rule, ADMET analysis, and molecular docking against Try-R and Try-S. Molecular dynamics studies were performed on selected compounds, and in vitro experiments were conducted to evaluate their activity against the promastigote and amastigote forms of L. donovani., Results: The virtual screening and subsequent analysis identified 33 promising compounds. Molecular dynamics studies of two compounds (comp-1 and comp-2) demonstrated stable binding interactions with the target enzymes and high affinity. In vitro experiments revealed that 13 compounds exhibited moderate activity against both the promastigote (IC 50 , 41 µM-76 µM) and the amastigote (IC 50 , 44 µM-72 µM) forms of L. donovani. Compounds 1 and 2 showed the highest percent inhibition and the lowest IC 50 values., Conclusion: The identified compounds demonstrated significant inhibitory activity against Leishmania donovani and stable interactions with target enzymes. These findings suggest that the compounds could serve as promising leads for developing new treatments for leishmaniasis., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

As part of our continuous research for the discovery of bioactive compounds against Trypanosoma cruzi and Leishmania infantum, the alkaloid (6aS)-dicentrine (1) was oxidized to afford (6aS,6S)- (2) and (6aS,6R)- (3) dicentrine-N-oxides. Evaluation of the cytotoxicity against NCTC cells indicated that 2 and 3 are non-toxic (CC 50 >200 μM) whereas 1 demonstrated CC 50 of 52.0 μM. Concerning T. cruzi activity against amastigotes, derivatives 2 and 3 exhibited EC 50 values of 9.9 μM (SI>20.2) and 27.5 μM (SI>7.3), respectively, but 1 is inactive (EC 50 >100 μM). Otherwise, when tested against L. infantum amastigotes, 1 and 3 exhibited EC 50 values of 10.3 μM (SI=5.0) and 12.7 μM (SI>15.7), respectively, being 2 inactive (EC 50 >100 μM). Comparing the effects of positive controls benznidazol (EC 50 =6.5 μM and SI>30.7) and miltefosine (EC 50 =10.2 μM and SI=15.2), it was observed a selective antiparasitic activity to diastereomers 2 and 3 against T. cruzi and L. infantum. Considering stereochemical aspects, it was suggested that the configuration of the new stereocenter formed after oxidation of 1 played an important role in the bioactivity against amastigotes of both tested parasites., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Examining host-pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host-parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host-parasite interaction., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Owing to the persistent number of parasitic deaths, Visceral leishmaniasis continues to haunt several economically weaker sections of India. The disease causes over 30,000 deaths and threatens millions annually on a global scale. The standard pentavalent antimonials, on the other hand, are associated with health adversities and disease relapse. The current study is focused on the search for the most potential natural bioactive phytocompound from the bark extract of the Northeastern Indian plant, Garcinia cowa, that shows potent anti-leishmanial properties. The High Resonance Liquid Chromatography followed by Mass Spectrometry (HR-LCMS) study followed by an in silico molecular docking using computational tools revealed that α-mangostin might potentially possess antiparasitic activity. To validate the anti-leishmanial efficacy of the compound, a cell viability assay was performed, which demonstrated the parasite-specific inhibitory activity of α-mangostin; with IC 50 values ranging from 4.95 - 7.37 µM against the different forms of Leishmania donovani parasite. The flow cytometric analysis of the phytocompound treated parasites indicated an oxidative and nitrosative stress-mediated apoptotic cell death in the parasites, by the suggestive surge in nuclear fragmentation and mitochondrial dysfunction. Simultaneously, a cytokine profiling study suggested approximate two-to-three-fold upregulated levels of pro-inflammatory cytokines post-compound treatment, which is predicted to actively contribute to parasite-killing. α-mangostin was also found to reduce the chances of parasite survival by inhibiting arginase enzyme activity, which in favorable conditions facilitates its sustenance. This study thereby substantiates that α-mangostin significantly possesses anti-leishmanial potentiality that can be developed into a cure for this infectious disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In the pursuit of developing novel anti-leishmanial agents, we conducted an extensive computational study to screen inhibitors from the FDA-approved ZINC database against Leishmania donovani glutathione synthetase. The three-dimensional structure of Leishmania donovani glutathione synthetase was constructed by homology modeling, using the crystallographic structure of Trypanosoma brucei glutathione synthetase as a template. Subsequently, molecular docking studies were carried out for a large number of compounds using AutoDock Vina. Among the screened compounds, we selected the top five with strong binding affinity to Leishmania donovani glutathione synthetase but having a very low affinity to its human homolog. Further investigations on protein-ligand complexes were done by conducting molecular dynamics (MD) simulation and MM/PBSA analysis. The results revealed that Olysio (Simeprevir) exhibited the lowest binding energy (-89.21 kcal/mol), followed by Telithromycin (-45.34 kcal/mol). These findings showed that these compounds have the potential to act as inhibitors of glutathione synthetase. Hence, our study provides valuable insights for the development of a novel therapeutic strategy against Leishmania donovani by targeting the glutathione synthetase enzyme.Communicated by Ramaswamy H. Sarma.

Leishmaniases comprise a group of infectious parasitic diseases caused by various species of Leishmania and are considered a significant public health problem worldwide. Only a few medications, including miltefosine, amphotericin B, and meglumine antimonate, are used in current therapy. These medications are associated with severe side effects, low efficacy, high cost, and the need for hospital support. Additionally, there have been occurrences of drug resistance. Additionally, only a limited number of drugs, such as meglumine antimonate, amphotericin B, and miltefosine, are available, all of which are associated with severe side effects. In this context, the need for new effective drugs with fewer adverse effects is evident. Therefore, this study investigated the anti-Leishmania activity of a dichloromethane fraction (DCMF) extracted from Arrabidaea brachypoda roots. This fraction inhibited the viability of L. infantum, L. braziliensis, and L. Mexicana promastigotes, with IC 50 values of 10.13, 11.44, and 11.16 µg/mL, respectively, and against L. infantum amastigotes (IC 50 = 4.81 µg/mL). Moreover, the DCMF exhibited moderate cytotoxicity (CC 50 = 25.15) towards RAW264.7 macrophages, with a selectivity index (SI) of 5.2. Notably, the DCMF caused damage to the macrophage genome only at 40 µg/mL, which is greater than the IC 50 found for all Leishmania species. The results suggest that DCMF demonstrates similar antileishmanial effectiveness to isolated brachydin B, without causing genotoxic effects on mammalian cells. This finding is crucial because the isolation of the compounds relies on several steps and is very costly while obtaining the DCMF fraction is a simple and cost-effective process. Furthermore, In addition, the potential mechanisms of action of brachydins were also investigated. The computational analysis indicates that brachydin compounds bind to the Triosephosphate isomerase (TIM) enzyme via two main mechanisms: destabilizing the interface between the homodimers and interacting with catalytic residues situated at the site of binding. Based on all the results, DCMF exhibits promise as a therapeutic agent for leishmaniasis due to its significantly reduced toxicity in comparison to the adverse effects associated with current reference treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leishmaniasis is a relevant disease worldwide due to its presence in many countries and an estimated prevalence of 10 million people. The causative agent of this disease is the obligate intracellular parasite Leishmania which can infect different cell types. Part of its success depends on its ability to evade host defense mechanisms such as apoptosis. Apoptosis is a finely programmed process of cell death in which cells silently dismantle and actively participate in several processes such as immune response, differentiation, and cell growth. Leishmania has the ability to delay its initiation to persist in the cell. It has been well documented that different Leishmania species target different pathways that lead to apoptosis of cells such as macrophages, neutrophils, and dendritic cells. In many cases, the observed anti-apoptotic effect has been associated with a significant reduction in caspase-3 activity. Leishmania has also been shown to target several pathways involved in apoptosis such as MAPK, PI3K/Akt, and the antiapoptotic protein Bcl-xL. Understanding the strategies used by Leishmania to subvert the defense mechanisms of host cells, particularly apoptosis, is very relevant for the development of therapies and vaccines. In recent years, the drug artemisinin has been shown to be effective against several parasitic diseases. Its role against Leishmania may be promising. In this review, we provide important aspects of the disease, the strategies used by the parasite to suppress apoptosis, and the role of artemisinin in Leishmania infection., Competing Interests: Conflicts of Interest The authors declare no conflicts of interest., (Copyright © 2024 Instituto Mexicano del Seguro Social (IMSS). Published by Elsevier Inc. All rights reserved.)